Ovary is the main regulator for female mammalian reproductive
function, as it regulates follicle development and reproductive hormones
secretion and produces mature oocytes. Numerous studies have indicated
that, in female mammalian ovaries, more than 99% of developing follicles
undergo atresia [1-3], which is mainly due to the apoptosis and
autophagy of granulosa cells [4-6]. Reactive oxygen species (ROS),
including superoxide anion radicals ([O.sub.2.sup.-]), hydrogen peroxide
([H.sub.2][O.sub.2]), and hydroxyl radical ([sup.*]OH), are produced
from normal cellular metabolism process and some external factors such
as exposure to agents known to cause oxidative stress [7, 8].
Physiological levels of ROS play an important role in intracellular
signal transduction, follicle development, ovulation, and gene
expression [9, 10], while excessive ROS production leads to oxidative
stress, which damages intracellular DNA, biomembrane lipids, proteins,
and other macromolecules [11]. Accumulating evidence shows that
excessive ROS cause the initiation of granulosa apoptosis and lead to
antral follicle atresia [8, 12]. Furthermore, growing evidence
demonstrates that high levels of ROS are associated with ovarian
toxicity and result in the gradual loss of fertility [8, 13].

FoxO (Forkhead O), a subfamily of transcription factors, including
FoxO1, FoxO3, FoxO4, and FoxO6, regulates diverse cellular functions
such as differentiation, proliferation, metabolism, survival, and death
[14]. As a key member of this family, FoxO1 plays a critical regulator
role in normal development of ovarian follicles [15]. FoxO1 is highly
expressed in granulosa cells of growing follicles [16] and modulates
lipid and sterol biosynthesis [15]. In addition, its expression is also
regulated by reproductive hormone and growth factors such as
follicle-stimulating hormone (FSH) and insulin-like growth factor-I
(IGF-I), which causes FoxO1 phosphorylation and promotes its nuclear
exclusion via phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway
[17]. Recent studies have demonstrated that FoxO1 plays an important
role in the regulation of cell death caused by oxidative stress. For
instance, in neurons and cardiac myocytes, FoxO1 induces cell death via
the translocation from the cytoplasm to the nucleus when these cells
suffered from oxidative stress [18, 19].

Diquat is a contact bipyridyl herbicide and potent prooxidant that
has been widely used to induce oxidative stress in different animals and
cellular models [20, 21]. Diquat can utilize molecular oxygen to produce
superoxide anion radical, and subsequently hydrogen peroxide through
dismutation, thus leading to serious damage to cellular components,
including lipids, proteins, and nucleic acids [22].

Grape seed procyanidin extracts (GSPEs) derived from grape seeds
have been reported to possess a broad spectrum of pharmacological and
medicinal properties [23]. Dimeric procyanidin B2 is one of the most
important components of GSPE and is probably more powerful than other
polyphenols. Some studies have shown that GSPB2 exhibits protective
effects against stress, inflammation, and cardiovascular diseases [24,
25]. However, there are few studies regarding the protective effects of
GSPB2 on follicular granulosa cell apoptosis induced by oxidative
stress. Thus, in the present study we investigated the protective
effects of GSPB2 on granulosa cell apoptosis and explored the possible
underlying mechanism.

2.2. Animals and Treatment. Female ICR mice were obtained from
Experimental Animal Center of Henan Province, China. All animal
experiments were performed in accordance with the recommendations in the
Animal Care and Use Guidelines of the Animal Care Advisory Committee and
were approved by the Animal Care Committee on the Ethics of Animal
Experiments of Henan Province. All mice were acclimated for one week
prior to use and maintained in a controlled environment with free access
to water and food, under a 12 h light-dark cycle and at a constant
temperature of 23 [+ or -] 2[degrees]C. The mice were divided randomly
into four groups containing ten animals in each. Table 1 describes the
study protocols. Diquat was dissolved in normal saline and administered
intraperitoneally, whereas GSPB2 were dissolved in normal saline and
supplemented to mice by intragastric administration. At the end of the
experiment, all mice were anesthetized and sacrificed, and ovaries were
collected. Granulosa cells were collected from the left ovaries for
measurement of ROS levels and other assays. The right ovaries were fixed
and embedded in paraffin for apoptosis assay.

2.3. Cell Isolation, Culture, and Treatment. Mice were injected
intraperitoneally with PMSG (10 IU) to stimulate follicular development
and sacrificed 48 h later. Ovaries were obtained and transferred into
Petri dishes (35 x 15 mm) filled with DMEM: F12 and then punctured with
a 5-gauge needle under a surgical dissecting microscope to release
granulosa cells. Next, the cells were plated at a density of 1 x
[10.sup.6] cells/mL in six-well culture plates (2 mL per well) or
96-well culture plates (200 [micro]L per well) and cultured in DMEM/F-12
with 10% (v/v) fetal bovine serum and incubated at 37[degrees]C with 5%
C[O.sub.2]. Before the formal experiment, the cultured cells were
treated with a range of concentrations (from 50 [micro]M to 200
[micro]M) of [H.sub.2][O.sub.2] to oxidative damage and the medium was
refreshed every 3 h by adding the determined concentration of
[H.sub.2][O.sub.2]. After determining cell activity, intracellular ROS
levels, and apoptosis rates, the dose of 150 [micro]M [H.sub.2][O.sub.2]
was chosen as the optimum concentration in the subsequent experiments.
Likewise, various concentrations of GSPB2 (1 to 20 [micro]mol/L) were
screened for the optimum concentration under [H.sub.2][O.sub.2]-induced
stress. Based on cell viability, 10 [micro]mol/L GSPB2 was determined as
the optimum concentration in the later formal experiment.

2.4. Determination of Cell Viability. Cell viability was determined
by MTT method. Briefly, the cultured granulosa cells were treated with
various concentrations of [H.sub.2][O.sub.2] for 6 h, and the cultured
cells were pretreated with different concentrations of GSPB2 for 24 h
and then treatment with [H.sub.2][O.sub.2]. To test cell viability, 200
[micro]L DMEM/F12 medium containing 0.5 mg/mL MTT was added to per well
and incubated for a further 4 h. The medium was removed and replaced
with 150 [micro]L of DMSO. The absorbance was measured on microplate
reader at 570 nm. The percentage of living cells was calculated by the
ratio of optical density of the experimental wells to that of the normal
wells.

2.5. Measurement of Intracellular ROS. Intracellular ROS levels
were measured using the GENMED intracellular ROS red fluorescence
determination kit. This assay is based on the principle that, in the
presence of ROS, dihydroethidium bromide (DHE) is rapidly oxidized to
become highly fluorescent products. These procedures were performed
according to the manufacturer's instructions. Image J software was
used to analyze the optical density in each granulosa cell.

2.6. In Situ TUNEL Analysis of Apoptosis. Apoptosis of granulosa
cells was detected by terminal deoxynucleotidyl transferase-mediated
dUTP-biotin nick end labeling (TUNEL) assay using a kit (Roche Applied
Science). The detailed procedure was performed according to the
manufacturer's instructions. Laser-scanning confocal microscope
(Leica) was used to obtain fluorescence images. Six fields of each
coverslip were randomly selected for counting, and 100 cells were
randomly counted for each field of vision. The total apoptotic cell
number and the total cell number were counted for six fields of vision.
The apoptosis rate was then calculated.

2.7. Cell Transfection and Treatments. Plasmids encoding FoxO1
shRNA or scrambled oligonucleotides were ordered from Sangon (Shanghai,
China). The sequence of FoxO1 shRNA and control shRNA are given in Table
2. Knockdown of FoxO1 was performed by transfecting granulosa cells with
FoxO1 shRNA. Transient transfection was performed using Lipofectamine
2000 (Invitrogen), according to the manufacturer's instructions.
The transfection efficiency was confirmed by Western blot. Twenty-four
hours after transfection, cultured granulosa cells were treated with 150
[micro]M [H.sub.2][O.sub.2] for 6 hours. Total RNA and proteins were
collected and preserved at -80[degrees]C until further analysis.

2.8. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR).
Total RNA was extracted from granulosa cells using TRIzol reagent
(Invitrogen, Carlsbad, CA, USA) according to the manufacturer protocol.
First cDNA strand was synthesized using PrimeScript RT Master Mix
(Takara Bio, Inc., Shiga, Japan). Quantitative real-time PCR (qRT-PCR)
was conducted using a fast real-time PCR system (Roche LightCycler 480
system). Triplicate samples were assessed for each gene of interest, and
GAPDH was used as a control gene. Relative expression levels were
determined by the [2.sup.-[DELTA][DELTA]Ct] method. Sequences of primers
used for apoptosis related genes are listed in Table 3. Primer sequences
for autophagy-related genes were obtained from published literatures
[26, 27].

2.9. Western Blot Analysis. The cultured granulosa cells were lysed
with ice-cold radioimmunoprecipitation assay (RIPA) buffer. The
whole-cell lysates (20 mg/lane) were separated on sodium dodecyl sulfate
polyacrylamide gel electrophoresis and transferred to a polyvinylidene
difluoride (PVFD) membrane. After the nonspecific binding sites were
blocked with 5% skim milk, the membrane was treated with anti-LC3B
rabbit monoclonal antibody or anti-FoxO1 (diluted 1:1000) overnight at
4[degrees]C. The immunoreactive bands were demonstrated by incubation
with horseradish peroxidase-(HRP-) conjugated goat anti-rabbit IgG
(diluted 1:3000) at room temperature for 1.5 hours. Protein bands were
visualized by exposing to an enhanced chemiluminescence detection system
(LAS-4000 imager, Fujifilm, Tokyo, Japan). Densitometry analyses were
performed and the values for target proteins were normalized to
[beta]-actin as the endogenous control.

2.10. Immunofluorescence Staining. Granulosa cells were grown on
coverslips and processed following a standard protocol. Briefly, the
granulosa cells were treated with 3.7% paraformaldehyde, permeabilized
with 0.5% Triton X-100, and incubated with anti-FoxO1 antibody (1:150)
for 60 min at 25[degrees]C. Then the cells were incubated with second
antibody for 60 min. DAPI was used to visualize the nuclei.
Laser-scanning confocal microscope (Leica) was used to obtain
fluorescent images.

2.11. Immunohistochemistry. Paraffin-embedded whole ovarian
sections were deparaffinized and rehydrated. Antigen retrieval was
treated in 10 mM citric buffer. 3% [H.sub.2][O.sub.2] was used to reduce
endogenous peroxide. Nonspecific binding was blocked with 3% bovine
serum albumin for 30 minutes. After washing, sections were incubated
overnight at 4[degrees]C with anti-LC3B rabbit monoclonal antibody
(diluted 1: 300), followed by incubation with a biotinylated secondary
antibody for 1 hour at a dilution of 1: 500. The sections were
counterstained with hematoxylin, then dehydrated, and mounted. In the
negative control group, the anti-LC3B antibody was replaced with 1% BSA.

2.12. Statistical Analysis. Statistical analysis was performed
using the SPSS 16.0 software (SPSS Inc., Chicago, IL, USA). All values
are expressed as mean [+ or -] SEM. The statistical significance between
groups was analyzed by one-way ANOVA and a P value of < 0.05 was
considered significant. All experiments were repeated at least three
times.

3. Results

3.1. Effects of Diquat Alone or Combined with GSPB2 on ROS Levels
in Granulosa Cells. Previous studies have indicated that more than
24mg/kg body weight of diquat in mice can result in acute toxicity [28].
The antioxidative dose for the GSPB2 was chosen because this was the
most effective dose [24]. In the present study, chronic exposure to
diquat (8 mg/kg, twice a week for 2 weeks) significantly induced the
granulosa cell damage. The granulosa cells were collected by puncture of
the dominant ovarian follicle from the left ovaries of control and
treated mice. The levels of ROS in granulosa cells were examined by ROS
red fluorescence determination kit. After treatment with diquat for 14
days, the ROS levels were significantly elevated in diquat group as
compared to those of the control group (Figures 1(a) and 1(b)). This
change was prevented by prior and concurrent supplementation of GSPB2 in
diquat plus GSPB2 group. No significant differences were found between
the control group and control plus GSPB2 group.

3.2. Effects of Diquat Alone or Combined with GSPB2 on Apoptosis in
Antral Follicles. The right ovaries from control and treated animal were
fixed and embedded in paraffin for the TUNEL assay. The percentage of
follicular granulosa apoptosis and TUNEL-positive follicles in diquat
group was higher than the control group (Figures 2(a) and 2(b)).
Compared with diquat alone-treated mice, the percentage of follicular
granulosa apoptosis and TUNEL-positive follicles was significantly
reduced in diquat plus GSPB2 group. Quantitative PCR analysis of
apoptosis-related genes showed that the expression levels of
proapoptotic genes (Bim and caspase-3) and FoxO1 were significantly
increased and the ratio of Bcl-2 to Bax was significantly decreased in
granulosa cells from diquat group compared with the control group.
Compared with diquat alone-treated mice, the expression levels of
proapoptotic genes and FoxO1 were significantly decreased and the ratio
of Bcl-2 to Bax was significantly increased in diquat plus GSPB2 group
(Figures 2(c) and 2(d)).

3.3. Effects of Diquat Alone or Combined with GSPB2 on Autophagy in
Antral Follicles. Granulosa cells were collected from the left ovaries
for quantitative PCR analysis. The right ovaries were fixed in 4%
paraformaldehyde for immunohistochemical analysis. The LC3 protein
levels were significantly enhanced in granulosa cells from diquat plus
GSPB2 group compared with the diquat alone-treated group (Figure 3(a)).
Consistent with the data from qRT-PCR assays, the relative mRNA
expression of some key autophagy genes (Lc3, Vps34, Atg12, and Beclin)
was significantly increased in granulosa cells from diquat plus GSPB2
treated mice compared with the diquat alone-treated mice (Figure 3(b)).

3.4. Effects of Diquat Alone or Combined with GSPB2 on Activities
of Antioxidant Enzymes and MDA Content in Ovarian Tissue. Ovaries from
control and treated mice were collected and homogenized for the
measurement of antioxidant enzymes and MDA content assay. We examined
the activities of T-SOD, CAT, and GSH-Px as well as MDA content in
ovarian tissues. The results showed that the activities of antioxidant
enzymes (T-SOD, GPx, and CAT) were significantly decreased and the MDA
contents were significantly increased in the ovary treated with diquat
as compared to control group (Figures 4(a)-4(d)). This variation trend
was attenuated in the ovary from mice treated with diquat plus GSPB2 as
compared to diquat alone-treated mice.

3.5. [H.sub.2] [O.sub.2] Induced Apoptosis in Cultured Granulosa
Cells. To further investigate the protective effects of GSPB2 and
underlying molecular mechanism on granulosa cells, we had primary
cultured granulosa cells exposed to [H.sub.2] [O.sub.2] and examined
their effects on cell viability. Cell viability was detected after
treatment with a range of concentrations (from 50 [micro]M to 200
[micro]M) of [H.sub.2][O.sub.2] and found that [H.sub.2][O.sub.2]
significantly reduced cell viability in a dose-dependent manner. When
the concentration of [H.sub.2][O.sub.2] reached 150 [micro]M, the cell
viability was significantly reduced with about 50% disruption of cells
as compared with that of control group (Figure 5(a)).

Moreover, treatment with 150 [micro]M [H.sub.2][O.sub.2] increased
significantly the intracellular ROS levels (Figures 5(b) and 5(c)) and
apoptosis rates in cultured cells (Figures 5(d) and 5(e)) compared with
those of control group, so the dose of 150 [micro]M [H.sub.2] [O.sub.2]
was chosen as the optimal concentration for the subsequent formal trial.
The results of ROS levels are shown in Figures 5(b) and 5(c); the ROS
levels are increased in a dose-dependent manner when exposed to
[H.sub.2][O.sub.2] for 6h. To evaluate the effects of ROS accumulation
on apoptosis, the cultured granulosa cells were treated with
[H.sub.2][O.sub.2] in various concentrations as indicated for 6h
(Figures 5(d) and 5(e)). The number of TUNEL-positive cells appeared to
increase dramatically and dose dependently. According to the results,
150 [micro]M [H.sub.2][O.sub.2] was chosen as the optimal concentration
for inducing granulosa cells oxidative stress in the subsequent
experiments.

3.6. GSPB2 Protected Granulosa Cells from
[H.sub.2][O.sub.2]-Induced Apoptosis. To evaluate whether GSPB2 protects
granulosa cells from oxidative stress, we used [H.sub.2][O.sub.2]
treatment in primary cultured cells and MTT assay was used to determine
cell viability (Figure 6(a)). As illustrated, the cells treated with 150
[micro]M [H.sub.2][O.sub.2] for 6h showed significant reduction in
viability compared to the control group. Pretreatment with the GSPB2
(10, 15, and 30 [micro]mol/L) significantly prevented
[H.sub.2][O.sub.2]-dependent damage and increased cell viability
(Figures 6(b) and 6(c)) shows intracellular ROS levels after incubation
with GSPB2 (10 [micro]mol/L). The ROS levels are decreased significantly
after GSPB2 treatment as compared with those of
[H.sub.2][O.sub.2]-treated group. Based on the TUNEL assay and DAPI
staining, the number of TUNEL-positive granulosa cells from
GSPB2-pretreated groups showed significant reduction as compared with
that in [H.sub.2][O.sub.2]-treated group (Figures 6(d) and 6(e)).
qRT-PCR was performed to detect the mRNA expression levels of key
apoptotic genes such as caspase-3, Bim, Bax, and Bcl-2 (Figure 6(f)).
Results showed that the expression levels of caspase-3 and Bim
significantly decreased, whereas the ratio of Bcl-2/Bax significantly
increased in the cells pretreated with GSPB2 as compared with those in
[H.sub.2][O.sub.2]-treated group.

3.7. GSPB2 Promoted Autophagy in Cultured Granulosa Cells. To
detect whether pretreatment with GSPB2 (10 [micro]mol/L) and exposure to
150 [micro]M [H.sub.2][O.sub.2] induced autophagy in cultured granulosa
cells, we examined mRNA levels of key autophagy genes (Vps34, Atg12,
Lc3b, and Beclin) and protein expression levels of LC3B. As shown in
Figure 7(a), the mRNA levels of some key autophagy genes were increased
significantly in the cells pretreated with GSPB2 as compared with those
in [H.sub.2][O.sub.2]-treated group. As predicted, the ratio of
LC3-II/[beta]-actin was significantly increased in the GSPB2 plus
[H.sub.2][O.sub.2] group as compared to that in
[H.sub.2][O.sub.2]-treated group (Figures 7(b) and 7(c)). Together,
these results demonstrate that autophagy is activated in the granulosa
cells treated with GSPB2.

3.8. GSPB2 Prevented FoxO1 Expression and Nuclear Localization in
Granulosa Cells. The effects of GSPB2 on FoxO1 expression levels in
cultured granulosa cells were measured with qRT-PCR and Western
blotting. The FoxO1 mRNA and protein levels were significantly elevated
in the [H.sub.2][O.sub.2]-treated granulosa cells as compared with those
in control group, whereas the FoxO1 mRNA and protein levels were
significantly reduced in the granulosa cells pretreated with GSPB2
compared to [H.sub.2][O.sub.2] alone (Figures 8(a)-8(c)).
Immunofluorescence studies indicated that [H.sub.2][O.sub.2]-induced
oxidative stress leads to FoxO1 translocation from the cytoplasm to the
nucleus, triggering predominant nucleus localization of FoxO1 as
compared to the control group. Compared with the
[H.sub.2][O.sub.2]-induced stress group, the predominant nucleus
localization of FoxO1 induced by [H.sub.2][O.sub.2] was suppressed in
the granulosa cells pretreated with GSPB2 (Figures 8(d) and 8(e)).

3.9. Knockdown of FoxO1 Inhibited GSPB2-Induced Autophagy. To
detect whether GSPB2-induced protection is dependent on autophagy, we
investigated whether the prevention of autophagy via 3-methyladenine
(3-MA) or the shFoxO1 plasmid influenced GSPB2-induced protection in
cultured granulosa cells. Cultured granulosa cells were pretreated with
GSPB2 for 24 h and then some were transfected with FoxO1 shRNA plasmid
or scrambled control shRNA plasmid for 24 h, and the others received 10
mmol/L of 3-MA, and finally exposure to 150 [micro]M [H.sub.2][O.sub.2]
for 6h. In shScramble (sh-Scr) group treated with GSPB2 (not with 3-MA),
the cell viability was significantly recovered in
[H.sub.2][O.sub.2]-induced stress group in contrast with the control
group. However, in shFoxO1 and sh-Scr treated with 3-MA groups, the same
treatment did not show increased cell viability (Figure 9(a)). To
further verify this result, we determined the expression levels of
caspase-3 mRNA and LC3B protein in these groups. The expression level of
caspase-3 significantly increased in the shFoxO1 and 3-MA groups (Figure
9(b)). Furthermore, GSPB2 induced the improvement of LC3-II protein in
the cells transfected with sh-Scr vector, whereas the other groups did
not increase in this protein level (Figures 9(c) and 9(d)). We next
evaluated the effects of FoxO1 knockdown or 3-MA supplement on apoptosis
in [H.sub.2][O.sub.2]-induced stress process. TUNEL staining results
indicated that shFoxO1 and 3-MA groups had a significant increase in
apoptosis rates, regardless of the supplement of GSPB2 (Figures 9(e) and
9(f)). Together, these results show that inhibiting autophagic process
or FoxO1 knockdown in granulosa cells can prevent the protective effects
of GSPB2.

4. Discussion

Oxidative stress affects multiple female reproductive processes
from ovarian follicular development to oocyte maturation, fertilization,
embryo development, and pregnancy [13]. In addition, numerous studies
have shown that oxidative stress plays a central role in physiological
and pathological processes of infertility and assisted fertility, normal
cycling ovaries, and cyclical endometrial changes [29]. In vitro and in
vivo supplementation of antioxidants is a strategy for overcoming
oxidative stress and enhancing female fertility [13, 30]. GSPB2 is one
of the main components of procyanidin extracts and has been reported to
possess more potent antioxidant and anti-inflammation properties greater
than B1, B4, and B5 [31]. Recent studies have shown that GSPB2 can
prevent AGEs (advanced glycation end products) induced ROS generation
and inhibit the human umbilical vein endothelial cell (HUVEC) apoptosis
[32, 33]. In the present study, we investigated the effect of GSPB2 on
[H.sub.2][O.sub.2]-induced granulosa cell apoptosis and found that GSPB2
significantly inhibited the cultured granulosa cell apoptosis by
downregulating FoxO1 expression in mRNA and protein levels. Moreover,
GSPB2 significantly reduced intracellular ROS production in
[H.sub.2][O.sub.2]-treated cells. The reduction in ROS production might
be related to a more direct role of the GSPB2 in the rescue of granulosa
cell apoptosis induced by oxidative stress. In our present in vivo and
in vitro studies, for the first time, we found that GSPB2 protected
follicular granulosa cell survival from oxidative stress via triggering
autophagic process. As previously reported, basal levels of autophagy
are important in maintaining cellular homeostasis by efficient removal
and recycling of damaged organelles and protein aggregates [34]. Our
results showed that GSPB2 supplementation enhanced antioxidant ability
and decreased oxidative stress and that GSPB2 reduced apoptosis and
increased autophagy caused by [H.sub.2][O.sub.2]-induced stress.

In female mammalian ovaries, more than 99% of the growing follicles
undergo atresia and degeneration during follicular growth and
development [1]. Recent studies have suggested that apoptosis and
autophagy are involved in the regulation of granulosa cell death during
ovarian follicular development and atresia [6, 35]. Furthermore,
numerous studies have demonstrated that autophagy can be triggered by
various stimuli that induce apoptosis, particularly oxidative stress
[36, 37]. Many animal and human studies have demonstrated that ROS exist
in the female reproductive tract, including ovaries and embryos [8, 38].
ROS are involved in the modulation of multiple physiological
reproductive functions such as oocyte maturation, ovarian
steroidogenesis, female fertility disorders, and the age-related decline
in fertility [39]. Diquat has been widely used as a contact herbicide
for the control of aquatic weeds and broad-leaved weeds among fruit,
vegetables, and other crops [40]. Diquat is a redox cycling agent that
generates ROS in cells. Thus, in this study, we investigated the toxic
effects of diquat on ovarian oxidative damage and the protective role of
GSPB2 against diquat-induced oxidative stress in ovaries. Our results
showed that ROS levels are significantly increased in follicular
granulosa cells (Figures 1(a) and 1(b)) and ovarian tissues of mice
exposed to diquat. Furthermore, significant increases in granulosa cell
apoptosis and TUNEL-positive follicles were also observed after diquat
treatment (Figures 2(a) and 2(b)). Our results suggested that chronic
exposure to diquat had toxic effects on the ovaries via enhancing ROS
production and inducing oxidative damage and that GSPB2 treatment
enhanced antioxidant ability and reduced oxidative stress.

In the female mammalian ovary, FoxO1 is highly expressed in
follicular granulosa cells of growing follicles [16]. Majority of
granulosa cells become apoptotic when growing follicles undergo atresia
and degeneration, and FoxO1 has been reported to play a pivotal role in
this process [15, 41]. FoxO1 is actively involved in the process of
proapoptosis via a mitochondria-independent and mitochondria-dependent
manner [42]. Moreover, FoxO1 activity can be regulated by in vivo and in
vitro growth factors and stress factors [43]. In response to growth
factors such as FSH, IGF-1, glucose, and insulin, protein kinase B (Akt)
and extracellular signal-regulated kinase (ERK) can phosphorylate FoxO1,
inducing FoxO1 interaction with chaperone proteins 14-3-3 and causing
its cytoplasmic retention [41, 44, 45]. In response to oxidative stress,
c-Jun N-terminal protein kinases (JNK), known as stress-activated
protein kinases, are activated by oxidative stress [46, 47]. Activated
JNK may phosphorylate 14-3-3 and interfere with 14-3-3/FoxO1 binding,
leading to FoxO1 release from its interaction with 14-3-3 and promoting
its nuclear translocation, thereby resulting in proapoptotic signaling
via induction of the transcription of proapoptotic target genes [48-50].
Our results showed that, after treatment with 150 [micro]M
[H.sub.2][O.sub.2], the FoxO1 was localized to the nuclei. Moreover,
GSPB2 supplement partly reverses this change via increasing the FoxO1
level in the cytoplasm (Figures 8(d) and 8(e)) and improves cell
viability by inhibiting apoptosis and inducing autophagic process.
Furthermore, GSPB2 induced a beneficial autophagic process that was
prevented when shFoxO1 was transfected to cultured granulosa cells, with
the same result as the group exposed to 3-MA. The GSPB2-induced
autophagic process was inhibited in shFoxO1 and 3-MA groups.
Furthermore, shFoxO1 or 3-MA improved caspase-3 expression and reduced
LC3-II protein level (Figures 9(a)-9(e)).

In summary, this study, for the first time, indicates the potential
protective activity of GSPB2 from procyanidin extracts on oxidative
stress-induced granulosa cell apoptosis and also clarifies the potential
underlying molecular mechanism. Moreover, these findings give us a clue
that targeting key transcription factor may be a potential therapeutic
avenue for the treatment of oxidative stress-related female reproductive
system diseases.

http://dx.doi.org/10.1155/2016/6147345

Conflict of Interests

The authors declare that there is no conflict of interests
regarding the publication of this paper.

This work was supported by the Key Program for Science and
Technology Development of Henan (152102110126) and the
Science-Technology Foundation for Outstanding Young Scientists of Henan
Academy of Agricultural Sciences (2016YQ19).

Caption: Figure 3: Effects of diquat alone or combined with GSPB2
on autophagy levels in antral follicles. Mice were treated with diquat
or saline or in combination with GSPB2. The right ovarian sections were
used for immunohistological study and granulosa cells were isolated from
left ovaries to investigate the changes of autophagy-related gene
expression. (a) Immunostaining of ovary sections was detected by using
anti-LC3-II. (b) The relative expression levels of autophagy-related
genes. Results are expressed as means [+ or -] SEM, n = 6, and * P <
0.05 versus diquat alone.